313 PHT

Lab. No. 8

Gram’s Stain

Gram’s +ve Gram’s -ve

Cocci Bacilli Cocci Bacilli

AerobicAerobic, , non-fermentativenon-fermentative, , motilemotile, , oxidase-positiveoxidase-positive gram-negative bacilli.gram-negative bacilli.

Most Important SpeciesMost Important SpeciesP.aeruginosa P.aeruginosa

opportunistic pathogen opportunistic pathogen causes UTI, wound infections and causes UTI, wound infections and

otitis mediaotitis media

Pseudomonas

Microscopical examination:Microscopical examination:

(morphology)(morphology)

A) Gram’s Stain:A) Gram’s Stain:

Gram –ve Gram –ve

Non-sporeforming bacilli , Non-sporeforming bacilli ,

having single arrangementhaving single arrangement..

B) Examination of Motility:B) Examination of Motility:

Using the Using the “Hanging Drop technique”“Hanging Drop technique”

Pseudomonas is highly motile by means of Pseudomonas is highly motile by means of polar flagella.polar flagella.

Cultural Cultural characteristic:characteristic:

It grows on It grows on simple media.simple media.

It usually produces It usually produces exopigments.exopigments.

1) Growth on nutrient 1) Growth on nutrient agar:agar:

Its growth on nutrient agar Its growth on nutrient agar

showing showing greenish greenish discolourationdiscolouration

due to due to exopigmentexopigment production. production.

Cetrimide agar is a Cetrimide agar is a highly selective highly selective mediummedium for pseudomonas species due for pseudomonas species due to presences of to presences of cetrimide cetrimide which which inhibits the growth of other bacteria.inhibits the growth of other bacteria.

It contains also It contains also MgClMgCl22 & & KK22SoSo44 to to facilitate production of the facilitate production of the charactaristic charactaristic green pigment green pigment of of pseudomonas. pseudomonas.

2) Growth on Cetrimide Agar:2) Growth on Cetrimide Agar:

Principle:

Results:

Only Pseudomonas species can grow on cetrimide agar showing growth of pale colonies with diffusion of green pigmentation.

MacConkey’s agar is a selective and differential medium

selective medium for selective medium for enteric enteric gram –ve gram –ve bacteriabacteria ( (bile saltbile salt inhibit the growth of inhibit the growth of non enteric bacteria).non enteric bacteria).

Test sugar:Test sugar: lactose.lactose. pH indicator:pH indicator: neutral neutral

redred ( yellow in alkaline, ( yellow in alkaline, pink in acidic pH).pink in acidic pH).

3) Growth on MacConkey’s agar:3) Growth on MacConkey’s agar:

Principle:

Gram –ve bacteriaGram –ve bacteria are classified into:are classified into:

Lactose fermenter (Pink colonies)

Lactose non-fermenter (pale colonies)

Results:

Pink colonies

Lactose fermenter

Pale coloniesLactose nonfermenter

Biochemical reaction:Biochemical reaction:

1)Oxidase test.1)Oxidase test.

2) Nitrate Test.2) Nitrate Test.

3) Oxidation Fermentation (O/F) Test.3) Oxidation Fermentation (O/F) Test.4) Growth on Triple Sugar Iron (TSI)

agar.

Results:

+ve Test: Appearance of purple colour within few seconds.

purple colour

+ve testPseudomonas

No colour

-ve testEnterobacteriaceae

1)Oxidase test:1)Oxidase test:

2) Nitrate Test:2) Nitrate Test:

Principle:

NitrateNitrate reductase

nitrite

α-naphthyl amine (nit. A)

Sulphanilic acid (nit. B)

Red diazonium saltEnterobacteriaceaeEnterobacteriaceae

Further reduction

Nirtogen (N2)

Add zinc dust (reducing agent)

If no red colour!

Procedure:

Nitrate broth

test m.o

Nit.A

Nit. B

Red colour

No red colour

Add zinc dust

Incubate at 35oC for 24

hrs

Results:

Red colour after addition of nit.A & nit.B

Reduction of Nitrate to nitrite

EnterobacteriaceaEnterobacteriaceaee

Red colour after addition of zinc dust

-ve reduction

No red colour after addition of zinc dust

Further reduction to

NitrogenPseudomonas

3) Oxidation Fermentation 3) Oxidation Fermentation (O/F) Test:(O/F) Test:

Sensitive O/F testSensitive O/F test

Positive Test:

O-/F- O+/F+ O+/F- O-/F+

Results:

Fermentative

EnterobacteriaceEnterobacteriaceaeae

Oxidative

Pseudomonas

Non

Saccharolytic

Principle:

butt slant

4) Growth on Triple Sugar Iron(TSI) agar:

Results:

1. No Fermentation:1. No Fermentation:

Butt: alkaline (red) Slant: alkaline (red)

2. Dextrose Fermentation:2. Dextrose Fermentation:

a) Initial reaction:

Dextrose

acid

(after 10 – 12 hrs)

Butt: acidic (yellow) Slant: acidic (yellow)

2. Dextrose Fermentation:2. Dextrose Fermentation:

b) Delayed reaction:(after 24 hrs)

Peptone O

2

Alkaline amines

Butt: acidic (yellow) Slant: alkaline (red)

Small amount of acid

3. Lactose Fermentation:3. Lactose Fermentation:

Lactose

Large amount of acid

Peptone O

2

Alkaline amines

Butt: acidic (yellow) Slant: acidic (yellow)

Results:

Butt:

Slant:

H2S :

acidic (yellow)

acidic (yellow)

-ve

acidic (yellow)

alkaline (red)

-ve

acidic (yellow)

alkaline (red)

+ve

alkaline (red)

alkaline (red)

-ve

Pseudomonas aeruginosa

I'm veryI'm very resistant to resistant to

most most antibioticsantibiotics, , so it's very so it's very hard to get hard to get

rid me. rid me.

Thank youThank you